164 Planta Medica 56(1990)
Antiplatelet Components in Panax ginseng Sheng-ChuKuo14, Che-Ming Teng2, Jang-ChangLee', Feng-Nien Ko2. Sheng-Chih Chen',
and Tian-Shung Wu3 Graduate Institute of Pharmaceutical Chemistry, China Medical College, 91 Hsueh Shih Road, Taichung, Taiwan, Republic of China 2 Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, Republic of China
Address for correspondence
Abstract Panaxynol and ginsenosides Ho, Hg1, and Rg2 were found to be the main antiplatelet components in
the diethyl ether and 1-butanol fractions, respectively, during the activity-guided fractionation of Panax ginseng, Panaxynol inhibited the aggregation, release reac-
tion, and thromboxane formation in rabbit platelets while ginsenosides Ho, Hg1, and Rg2 suppressed the release reaction only.
Key words Panaxynol, ginsenosides, antiplatelet activity, aggregation activity, ATP release, thromboxane formation.
7.8 mm; from Macherey-Nagel, West Germany) with n-hexanedichloromethane (3: 1) as the eluent.
Reagents and solutions Reagents and solvents for extraction, chemical analysis, and HPLC were purchased from E. Merck (Darmstadt, West Germany), and were used as received. Thin-layer chromatography for fractionation was performed on precoated aluminum TLC sheets (silica gel 60, 1mm layer; from E. Merck) with petroleum ether-diethyl ether-acetic acid (100: 15: 1) as a developer.
Collagen (Type 1, bovine achilles tendon; from Sigma Chem. Co.) was homogenized in 25mM acetic acid and stored at —70°C at a concentration of 1 mg/mI. Thrombin (bovine)
was purchased from Parke Davis & Co. and dissolved in 50% glycerol to give a stock solution of 100 NIH units/mi. lonophore A23187 was obtained from Calbiochem-Behring Co. and dissolved in dimethyl sulfoxide (DMSO). Platelet-activating factor (PAF) was
also a product of Calbiochem-Behring Co. and dissolved in
Introduction Panax ginseng C. A. Meyer is used as a panacea and is one of the most important oriental medicines
(1). Recent studies indicated that ginsenosides isolated from P. ginseng improved blood lipid metabolism and thus prevented atherosclerosis (2). Matsuda et al. also indicated that a 70% methanol extract and ginseno side Ro retarded
blood coagulation and enhanced fibrinolysis (3—6). Nakanishi also reported the inhibitory effects of ginsenosides on platelet functions (7). However, the results varied and were not able to support the clinical investigation on the activity of P. ginseng. For these reasons, the following study was carried out to find the most active antiplatelet components in P. ginseng.
Materials and Methods Apparatus
A Shimadzu IR-400 spectrophotometer, a Bruker AC-250 NMR spectrometer with TMS as an internal reference, and
a Hewlett-Packard 5995 GC-MS system were used for the spectrometric identification of the isolated compounds. The isolation of
panaxynol from GSE-5 was carried out on a Hewlett-Packard 1084B liquid chromatograph equipped with a Hewlett-Packard 79850B LC terminal and a Nucleosil 1OCN column (5itm, 300 x
chloroform. AD?, arachidonic acid, fibrinogen (rabbit, 90% clottable), EDTA (disodium salt), bovine serum albumin, apyrase, and luciferase-luciferin were purchased from Sigma Chem. Co.
Fractionation and isolation of the antiplatelet components A procedure for the preparation of the fractions and GSE-5 was partially adopted from the previous papers (8, 9), and is given in Chart 1. The fractions thus obtained were tested for their antiplatelet activity. From the elute of the HPLC separation of GSE-5, panaxynol was obtained and identified by comparison with the IR, 1H-NMR, 13CNMR and mass spectra reported previously (10—12). On the other hand, Ro, Hg1 and Rg2 were also isolated and identified with the help of the procedure described by Sanada and co-workers (8) from the fraction D for the reinvestigation of their inhibitory activity.
Preparation of platelet suspension (13, 14) Blood was collected from the rabbit marginal ear vein and was mixed with EDTA to a final concentration of 6 mM. It
was centrifuged for 10mm at 90 x g and room temperature, and the supernatant was obtained as a platelet-rich plasma. the latter was further centrifuged at 500 x g for 10 mm. The platelet pellets were washed with Tyrode's solution (Ca2-free)/2 mM EDTA/
0.1mg mt1 apyrase/3.5mg• ml1 serum albumin, and centrifuged at 500 x g for 10 mm. Then the pellets were washed with
Downloaded by: National University of Singapore. Copyrighted material.
Received: October 25, 1988
Planta Medica 56(1990) 165
Antiplatelet Components in Panax ginseng water Panax ginseng powder
[F]
80°C MeOH, ref luxed
methanol extract
[A] suspended in water extracted with ether
water extract
ether extract
[B]
[C]
extracted with
Thin layer chromatography (silica gel; petroleum ether-diethyl etheracetic acid, 100:15:11
water-saturated 1-butanol
1-butanol extract
water extract
[D]
[E] Fr. 1
Fr. 2 Fr. 3
Fr. 6 Fr. 7
Fr. 4 Fr. 5
chromatography (CHCI3-MeOH-
TLC, 3 times
H20, 65:35:10(
Ro
CS [-5 Rb1
HPLC Rc
Rb2
Rf
Rg1
Rg2
Chart 1
panaxynol (1(
the above Tyrode's solution without EDTA. After centrifugation at
Table 1 The effect of the fractions of Panaxginsengon platelet aggregation
the same conditions, the platelet pellets were finally suspended in Tyrode's solution of the following composition (mM): NaCl (138.6),
induced by ADP, arachidonic acid, and collagen. Aggregation (% of control)
KCI (2.8), NaHCO3 (11.9), MgC12 (1.1), NaH2PO4 (0.33), CaC1 (1.0),
and glucose (11.2).
Fraction
Platelet aggregation andATP release reaction
A B
Aggregation was measured by a turbidimetric
C D
method (15, 16). ATP released from platelets was detected by the bioluminesence method as described by DeLuca and McElory (17). Both aggregation and release reaction were simultaneously and continuously measured by a Lumi-aggregometer (Model 1020,
Payton, Canada) connected to two dual-channel recorders. All glassware was siliconized. Just 1 mm before the addition of the
E
F
ADP
AA
Collagen
71 92 74
29 86 6
88 97
75 95 99
41 98 89
57 90 99 94
Platelets were incubated with each fraction (2 mg/mI) or 0.5% DMSO at 37°C for I mm, then ADP (20 xM(, arachidonic acid (AA, 100 sM(, or collagen (10 .g/mI( was added to trigger the aggregation. Means of 2 to 3 determinations are presented.
aggregation inducer, the platelet suspension was stirred at 900rpm. AT? of known concentration was used to calibrate the intensity of the bioluminescence. The percentage of aggregation was calculated as follows (A, absorbance):
[
initial A — final A after aggregation Aggregation (%) = _____________________________ initial A — A of
suspending medium
Results and Discussion The fractions prepared with the procedure given in Chart 1 have been tested for their antiplatelet activity. As can be seen from Table 1, fraction A was found to be slightly more effective than fraction D, indicating that the
enrichment of the fraction D in ginsenosides had not decreased the aggregation activity. Hence, it is clear that ginsenosides are not the main antiplatelet components in P. ginseng. In addition, the water extracts (fractions B, E, and F) show little effect on the platelets, suggesting that there is
no active component present in the fractions. Therefore, the most active antiplatelet component is not present in the fraction D but in fraction C. Accordingly, the fraction C was further fractionated into fractions 1—7.
1
Control
Jr lox
I Collagen
AA
ADP
[
Throinbin
PA F
A23187
Fig. 1 Effect of GSE-5 on the aggregation and AlP release of washed platelets. Platelets were incubated with GSE-5 (100 sg/m() or 0.5% DMSO (control) at 37°C for I mm, then collagen (10 xg/ml), arachidonic acid (AA, 100 .LM), ADP (20 sM), thrombin (0.1 U/mI), PAF (2 ng/ml), or ionophore A23 187 (2 .tM) were added to trigger the aggregation (upward tracings) and ATP release (downward tracings).
Downloaded by: National University of Singapore. Copyrighted material.
silica gel column
166 Planta Medica 56(1990)
Sheng-Chu Kuo et al. Table 2 Inhibitory effect of the fractions from the fraction C of Panaxginsengon platelet aggregation induced by ADP, arachidonic acid, collagen, and
Aggregation (%)
Fraction
ADP
Control 3 4 5 6 7
AA
77.9 76.7 77.5
3.6(4) 2.0(4)
18.9
9.5(4)"
67.1 39.7
7.1(4)
1.7(7)
12,9(4)'"
90.6 87.2 89.4 32.5 84.0 63.0
PAF
Collagen
3.5(4)
90.9 90.9 89.7 7.9 85.3
9.1(4)'"
77.1
2.7(6) 3.9(4)
3.8(4)
16.3(4)'"
platelet-activating factor (PAF).
1.1 (4)
98.3
0.7(6)
1.1(4)
95.1
2.9(4)
93.7 75.2 91.1
0.8(5)" 1.6(5)" 12.8(5)' 3.2(5)'
87.2
4.3 (5y'
6.8(4)" 4.6(4) 7.2(4)
Washed rabbit platelets were incubated with the test solution (100 gIml) at 37°C for 1 mm, the ADP (20 tM), arachidonic acid IAA, 100 .LMI, collagen (10 xg/ml) or PAF (2 ng/ml) were added to trigger the aggregation. Values are presented as means S.E. (n)': p
Antiplatelet Components in Panax ginseng Sheng-ChuKuo14, Che-Ming Teng2, Jang-ChangLee', Feng-Nien Ko2. Sheng-Chih Chen',
and Tian-Shung Wu3 Graduate Institute of Pharmaceutical Chemistry, China Medical College, 91 Hsueh Shih Road, Taichung, Taiwan, Republic of China 2 Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, Republic of China
Address for correspondence
Abstract Panaxynol and ginsenosides Ho, Hg1, and Rg2 were found to be the main antiplatelet components in
the diethyl ether and 1-butanol fractions, respectively, during the activity-guided fractionation of Panax ginseng, Panaxynol inhibited the aggregation, release reac-
tion, and thromboxane formation in rabbit platelets while ginsenosides Ho, Hg1, and Rg2 suppressed the release reaction only.
Key words Panaxynol, ginsenosides, antiplatelet activity, aggregation activity, ATP release, thromboxane formation.
7.8 mm; from Macherey-Nagel, West Germany) with n-hexanedichloromethane (3: 1) as the eluent.
Reagents and solutions Reagents and solvents for extraction, chemical analysis, and HPLC were purchased from E. Merck (Darmstadt, West Germany), and were used as received. Thin-layer chromatography for fractionation was performed on precoated aluminum TLC sheets (silica gel 60, 1mm layer; from E. Merck) with petroleum ether-diethyl ether-acetic acid (100: 15: 1) as a developer.
Collagen (Type 1, bovine achilles tendon; from Sigma Chem. Co.) was homogenized in 25mM acetic acid and stored at —70°C at a concentration of 1 mg/mI. Thrombin (bovine)
was purchased from Parke Davis & Co. and dissolved in 50% glycerol to give a stock solution of 100 NIH units/mi. lonophore A23187 was obtained from Calbiochem-Behring Co. and dissolved in dimethyl sulfoxide (DMSO). Platelet-activating factor (PAF) was
also a product of Calbiochem-Behring Co. and dissolved in
Introduction Panax ginseng C. A. Meyer is used as a panacea and is one of the most important oriental medicines
(1). Recent studies indicated that ginsenosides isolated from P. ginseng improved blood lipid metabolism and thus prevented atherosclerosis (2). Matsuda et al. also indicated that a 70% methanol extract and ginseno side Ro retarded
blood coagulation and enhanced fibrinolysis (3—6). Nakanishi also reported the inhibitory effects of ginsenosides on platelet functions (7). However, the results varied and were not able to support the clinical investigation on the activity of P. ginseng. For these reasons, the following study was carried out to find the most active antiplatelet components in P. ginseng.
Materials and Methods Apparatus
A Shimadzu IR-400 spectrophotometer, a Bruker AC-250 NMR spectrometer with TMS as an internal reference, and
a Hewlett-Packard 5995 GC-MS system were used for the spectrometric identification of the isolated compounds. The isolation of
panaxynol from GSE-5 was carried out on a Hewlett-Packard 1084B liquid chromatograph equipped with a Hewlett-Packard 79850B LC terminal and a Nucleosil 1OCN column (5itm, 300 x
chloroform. AD?, arachidonic acid, fibrinogen (rabbit, 90% clottable), EDTA (disodium salt), bovine serum albumin, apyrase, and luciferase-luciferin were purchased from Sigma Chem. Co.
Fractionation and isolation of the antiplatelet components A procedure for the preparation of the fractions and GSE-5 was partially adopted from the previous papers (8, 9), and is given in Chart 1. The fractions thus obtained were tested for their antiplatelet activity. From the elute of the HPLC separation of GSE-5, panaxynol was obtained and identified by comparison with the IR, 1H-NMR, 13CNMR and mass spectra reported previously (10—12). On the other hand, Ro, Hg1 and Rg2 were also isolated and identified with the help of the procedure described by Sanada and co-workers (8) from the fraction D for the reinvestigation of their inhibitory activity.
Preparation of platelet suspension (13, 14) Blood was collected from the rabbit marginal ear vein and was mixed with EDTA to a final concentration of 6 mM. It
was centrifuged for 10mm at 90 x g and room temperature, and the supernatant was obtained as a platelet-rich plasma. the latter was further centrifuged at 500 x g for 10 mm. The platelet pellets were washed with Tyrode's solution (Ca2-free)/2 mM EDTA/
0.1mg mt1 apyrase/3.5mg• ml1 serum albumin, and centrifuged at 500 x g for 10 mm. Then the pellets were washed with
Downloaded by: National University of Singapore. Copyrighted material.
Received: October 25, 1988
Planta Medica 56(1990) 165
Antiplatelet Components in Panax ginseng water Panax ginseng powder
[F]
80°C MeOH, ref luxed
methanol extract
[A] suspended in water extracted with ether
water extract
ether extract
[B]
[C]
extracted with
Thin layer chromatography (silica gel; petroleum ether-diethyl etheracetic acid, 100:15:11
water-saturated 1-butanol
1-butanol extract
water extract
[D]
[E] Fr. 1
Fr. 2 Fr. 3
Fr. 6 Fr. 7
Fr. 4 Fr. 5
chromatography (CHCI3-MeOH-
TLC, 3 times
H20, 65:35:10(
Ro
CS [-5 Rb1
HPLC Rc
Rb2
Rf
Rg1
Rg2
Chart 1
panaxynol (1(
the above Tyrode's solution without EDTA. After centrifugation at
Table 1 The effect of the fractions of Panaxginsengon platelet aggregation
the same conditions, the platelet pellets were finally suspended in Tyrode's solution of the following composition (mM): NaCl (138.6),
induced by ADP, arachidonic acid, and collagen. Aggregation (% of control)
KCI (2.8), NaHCO3 (11.9), MgC12 (1.1), NaH2PO4 (0.33), CaC1 (1.0),
and glucose (11.2).
Fraction
Platelet aggregation andATP release reaction
A B
Aggregation was measured by a turbidimetric
C D
method (15, 16). ATP released from platelets was detected by the bioluminesence method as described by DeLuca and McElory (17). Both aggregation and release reaction were simultaneously and continuously measured by a Lumi-aggregometer (Model 1020,
Payton, Canada) connected to two dual-channel recorders. All glassware was siliconized. Just 1 mm before the addition of the
E
F
ADP
AA
Collagen
71 92 74
29 86 6
88 97
75 95 99
41 98 89
57 90 99 94
Platelets were incubated with each fraction (2 mg/mI) or 0.5% DMSO at 37°C for I mm, then ADP (20 xM(, arachidonic acid (AA, 100 sM(, or collagen (10 .g/mI( was added to trigger the aggregation. Means of 2 to 3 determinations are presented.
aggregation inducer, the platelet suspension was stirred at 900rpm. AT? of known concentration was used to calibrate the intensity of the bioluminescence. The percentage of aggregation was calculated as follows (A, absorbance):
[
initial A — final A after aggregation Aggregation (%) = _____________________________ initial A — A of
suspending medium
Results and Discussion The fractions prepared with the procedure given in Chart 1 have been tested for their antiplatelet activity. As can be seen from Table 1, fraction A was found to be slightly more effective than fraction D, indicating that the
enrichment of the fraction D in ginsenosides had not decreased the aggregation activity. Hence, it is clear that ginsenosides are not the main antiplatelet components in P. ginseng. In addition, the water extracts (fractions B, E, and F) show little effect on the platelets, suggesting that there is
no active component present in the fractions. Therefore, the most active antiplatelet component is not present in the fraction D but in fraction C. Accordingly, the fraction C was further fractionated into fractions 1—7.
1
Control
Jr lox
I Collagen
AA
ADP
[
Throinbin
PA F
A23187
Fig. 1 Effect of GSE-5 on the aggregation and AlP release of washed platelets. Platelets were incubated with GSE-5 (100 sg/m() or 0.5% DMSO (control) at 37°C for I mm, then collagen (10 xg/ml), arachidonic acid (AA, 100 .LM), ADP (20 sM), thrombin (0.1 U/mI), PAF (2 ng/ml), or ionophore A23 187 (2 .tM) were added to trigger the aggregation (upward tracings) and ATP release (downward tracings).
Downloaded by: National University of Singapore. Copyrighted material.
silica gel column
166 Planta Medica 56(1990)
Sheng-Chu Kuo et al. Table 2 Inhibitory effect of the fractions from the fraction C of Panaxginsengon platelet aggregation induced by ADP, arachidonic acid, collagen, and
Aggregation (%)
Fraction
ADP
Control 3 4 5 6 7
AA
77.9 76.7 77.5
3.6(4) 2.0(4)
18.9
9.5(4)"
67.1 39.7
7.1(4)
1.7(7)
12,9(4)'"
90.6 87.2 89.4 32.5 84.0 63.0
PAF
Collagen
3.5(4)
90.9 90.9 89.7 7.9 85.3
9.1(4)'"
77.1
2.7(6) 3.9(4)
3.8(4)
16.3(4)'"
platelet-activating factor (PAF).
1.1 (4)
98.3
0.7(6)
1.1(4)
95.1
2.9(4)
93.7 75.2 91.1
0.8(5)" 1.6(5)" 12.8(5)' 3.2(5)'
87.2
4.3 (5y'
6.8(4)" 4.6(4) 7.2(4)
Washed rabbit platelets were incubated with the test solution (100 gIml) at 37°C for 1 mm, the ADP (20 tM), arachidonic acid IAA, 100 .LMI, collagen (10 xg/ml) or PAF (2 ng/ml) were added to trigger the aggregation. Values are presented as means S.E. (n)': p
